Selenophosphate is the active selenium donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although in some cases free selenide, a very toxic substance, can be used in vitro for synthesis of selenophosphate, a "selenane selenium" form of selenium designated Se-zero appears to function in vivo. This form of selenium is made available directly to selenophosphate synthetase by "selenium delivery proteins". The aim of the present study was to find types of selenium compounds that could be used as substrate by the "selenium delivery proteins". Selenotrisulfides, RSSeSR, are among the possible substrates being considered. Low molecular weight thiols and reactive thiol groups of proteins can interact with selenite to form RSSeSR derivatives. The derivative of glutathione is stable at acid pH values but rapidly decomposes at neutral to alkaline pH. Reaction of penicillamine (3-mercaptovaline) with selenite gave a derivative, PSSeSP, that was stable below pH 8.0 but gradually decomposed at neutral pH in the presence of excess PSH. The perselenide, PSSe, product appears to be very unstable and was not detected when PSSeSP was reduced. Attempts to form an adduct of the dithiol form of thioredoxin were unsuccessful. Upon addition of selenite to the reduced thioredoxin, the oxidized protein was formed at once and no Se-containing intermediate was found. A mammalian protein, rhodanese, that normally occurs as a persulfide of a critical cysteine residue is thought to serve as a sulfur delivery protein. This protein was reacted in its reduced form with selenite and GSH. A rhodanese derivative containing bound selenium was formed and this was stable at neutral pH in the presence of excess GSH at 37 degrees centigrade. Presumably, the selenium is present in the form of a perselenide derivative of a reactive cysteine in the protein instead of the usual persulfide. The possible physiological roles of the penicillamine and rhodanese selenium derivatives as selenium donor compounds are under investigation. A preliminary study indicated that the rhodanese-bound selenium could be utilized in the selenophosphate synthetase assay system. For certain selenoenzymes that contain a perselenide type of structure that is coordinated to molybdenum, the delivery proteins could serve as a direct selenium source.